As chemical and biological sample complexity has grown, analytical techniques have evolved to maintain accuracy and precision of final results. These techniques often include running multiple calibration curves throughout the day with multiple controls. One way to increase throughput and efficiency is to analyze multiple samples at the same time, which is typically referred to as multiplexing. Multiplexing generally may include the analysis of multiple samples simultaneously.
Sample throughput can be achieved using either parallel or serial multiplexing. In serial approaches, samples are run one after another. By decreasing the time of each individual analysis, the number of samples that can be analyzed is increased. In parallel approaches, multiple samples are run simultaneously. While the length of a single experiment may be longer than that for a serial approach, this can be offset by the ability to analyze multiple samples at one time.
Mass spectrometry (MS) typically achieves sample multiplexing through chemical tagging approaches using a variety of isotope labeling schemes. Using these approaches, parallel multiplexing of up to 10 samples may be possible, although often fewer samples are run simultaneously. These methods also provide advantages in quantitation because differences in ionization efficiency/signal response between samples can be normalized because they are analyzed simultaneously, which accounts for the matrix heterogeneity of each sample. Therefore, even though competing ionization and signal suppression may occur, these phenomena should be experienced equally by all of the samples.
These approaches, however, typically suffer from one or more disadvantages, such as the one or more difficulties associated with the labeling chemistry that is required to covalently attach a tag to the analytes. The reactions usually are directed to specific functional groups, but these specific functional groups may not be present on all compounds of interest. Tagging efficiencies also can vary between samples and/or between analytes within each sample. Another disadvantage is the cost of at least some of these reagents.
Frequency-modulation (FM) is one approach to multiplexing in which each sample may be encoded at a particular frequency. The total signal detected by the instrument may then be a sum of all the frequency components in the mixture, and deconvolution by Fourier transform (FT) can reveal its unique spectral components. Therefore, the identity of each sample may be encoded by its unique frequency and its concentration may be encoded by the peak height in the frequency domain. Besides the ability for parallel multiplexing, one possible advantage of this method is that it inherently filters the signal since the frequency of each component can be modulated away from noise and selected with appropriate bandwidths. Combined, these benefits often result in an increased S/N.
For optical measurements, FM multiplexing has been achieved by modulating the intensities of multiple light sources at unique frequencies. The emission from each compound can then be encoded at the frequency that was used to excite it. One demonstration of this method was used in the determination of multiple elements simultaneously using atomic absorption spectroscopy (see Edel H. et al., Anal. Bioanal. Chem. 1996; 355:292-4). In this approach, the power of three hollow cathode lamps and a deuterium lamp were modulated independently at unique frequencies. Lock-in amplifiers were used to monitor the signal from each source independently of the other sources and background emission from the furnace. This technique has been used to analyze 11 elements simultaneously. In another example of FM multiplexing, two lasers were pulsed at unique frequencies for simultaneous measurement of two DNA samples that were being separated by electrophoresis (see Dongre C, et al., Lab on a Chip. 2011; 11(4):679-83).
One technique particularly suited for analyzing multiple analytes in a single run is mass spectrometry (MS). One of the most common methods for multiplexing samples for analysis by MS is through incorporation of tags into one, or multiple, samples. For example, stable isotope labeling by amino acids in cell culture (SILAC), isotope coded affinity tags, and isobaric tags for relative and absolute quantification (iTRAQ™, Applied Biosystems, USA). iTRAQ™, which utilizes covalent labeling of primary amines with isobaric tags, can enable multiplexing and relative quantitation of up to 8 different samples.
Other techniques that utilize a frequency-based approach for increasing the throughput of a single sample and thereby increase S/N of the measurement include the following: FT-ion mobility spectroscopy (FT-IMS)(see Knorr, F. J. et al., Anal. Chem. 1985; 57(2):402-6), Hadamard Transform Time-of-Flight MS (HT TOF-MS) (see Fernandez, F. M. et al., Anal. Chem. 2002; 74(7):1611-7), Hadamard Transform capillary electrophoresis (HT-CE)(see Kaneta, T. et al., Analytical Chemistry 1999; 71(23):5444-6), Shah convolution FT detection (SCOFT) (see Kwok, Y. C. et al., J. Chromatogr. A. 2001; 924(1-2):177-86), FT-CE (see Allen, P B, et al., Analytical Chemistry. 2007; 79(17):6807-15), or a general fast Fourier transform separation (see Trudgett, M. J., et al., J. Chromatogr. A. 2011; 1218(22):3545-54). While each of these systems are unique in the way in which they utilize frequency-based approaches for increasing S/N, they are all configured to analyze a single sample.
Each of these methods suffers from one or more disadvantages, including, but not limited to, their cost, the diligence required to achieve similar labeling efficiencies, possible background interference, and the one or more inefficiencies associated with the ability to analyze a single sample.
There remains a need for a relatively inexpensive and/or non-labeling approach for parallel and/or serial multiplexing by MS. There also remains a need for methods that avoid or reduce background interference, eliminate or reduce one or more difficulties associated with achieving similar labeling efficiencies, analyze two or more samples, or a combination thereof.